• Biomass Pre-Treatment
    Analytical Methods

There has been a considerable focus in recent years on the production of advanced biofuels, also known as cellulosic biofuels, and biomass-derived chemicals in biorefining processes. These technologies use lignocellulosic biomass, which is mostly composed of the biopolymers of cellulose, hemicellulose and lignin. Lignocellulosic biomass is highly abundant and is typically of a significantly lower cost than the biomass (such as wheat, sugar beet and rapeseed) used to produce conventional biofuels.

The key to exploiting the chemical value of lignocellulosics is to depolymerise the lignocellulosic matrix in order to obtain smaller molecules that can be utilised, or further converted to platform chemicals and biofuels. There are two major pathways by which biorefineries undertake this conversion: through hydrolysis processes that aim to liberate sugars from the lignocellulosic polysaccharides (i.e. cellulose and hemicellulose), and through thermal processes (such as pyrolysis and gasification) that degrade more extensively the components of both polysaccharides and lignin.

Why Pre-treatment is Necessary

In the hydrolysis process cellulose and hemicellulose are hydrolysed (broken apart) in pure water through attack by the hydrogen atoms of the water molecule on these polysaccharides. This is a very slow reaction, particularly for cellulose due to its recalcitrance to hydrolysis, but it can be sped up using elevated temperatures and pressures, and catalysed by acids (concentrated or dilute) and highly selective enzymes such as cellulases. However, the rates of hydrolysis of cellulose when processing virgin biomass are still very low due to the recalcitrance of the lignocellulosic matrix. In particular, the complex inter-associations between hemicellulose and cellulose, the crystalline nature of much cellulose, and the existence of a physical barrier of lignin surrounding the cellulose fibres are said to be major hindrances.

Biomass is more than just cellulose, however, and the process is complicated by the relative ease with which hemicellulose can be hydrolysed meaning that a severe means of hydrolysis targeted for the liberation of glucose from cellulose may result in the sugars liberated from hemicellulose being degraded.

For these, and other, reasons most hydrolysis technologies involve a pretreatment step prior to the hydrolysis of the cellulose. This typically targets the removal of hemicellulose and/or lignin, leaving a solid predominately cellulosic residue that is more amenable to conversion. The hemicellulose is typically present in the liquid output from the pretreatment and can be valorised in separate processes.

Types of Pre-Treatments

There are large number of different pretreatment technologies that can be employed in biorefineries prior to the primary hydrolysis stage. One of the more common types is steam explosion which involves partially comminuted biomass being subjected to high pressure steam (at 210–290 C) for several minutes before this steam is rapidly vented, resulting in an explosive decompression and flash cooling of the biomass. Liquid Hot Water is another pretreatment process and involves superheated water (180–230 C) that is kept in the liquid state through high pressures and put in contact with the biomass via various pathways (including co-current, counter-current, and flow-through processes). The process will hydrolyse most of the hemicellulose and up to two thirds of the lignin and one quarter of the cellulose, depending on conditions. The use of dilute-acids is a more well-established pretreatment, whilst other pretreatments involve the use of alkalis and other solvents (e.g. ethanol, acetone etc.). There has also been much research recently on the use of ionic liquids as green solvents for biomass.

Important Considerations When Designing a Pretreatment

Particularly when elevated temperatures and/or chemicals are employed in pretreatment it can be possible for the production of the monosaccharide (e.g. from the hydrolysis of hemicellulose) to not be the end-point of the process. Some sugars may be further degraded to a variety of potential products. This is important since these products can often be inhibitory to fermentation or can otherwise complicate subsequent downstream processing methods. However, a number of these sugar degradation products (such as levulinic acid, furfural, and hydroxymethylfurfural) can be valuable chemicals in their own right and some technologies target their production.

However, in other cases the pretreatments may only partially hydrolyse some of the polysaccharides. This partial hydrolysis can result in a significant proportion of the hemicellulosic and/or cellulosic sugars being tied up in soluble oligosaccharides that are contained within the process liquor. It is important to know whether the soluble sugars are present in monosaccharide or oligomer forms as these will affect downstream processing and the effective valorisation of the liquid component.

Therefore, the compositions of the liquid and solid outputs of biomass pretreatment processes can form a wide spectrum of potential products that can exist in greatly varying concentrations which will depend on: the feedstock, the type of pretreatment, and the wide array of potential pretreatment conditions (e.g. solid loading, temperature, pressure, solvent etc.).

Recommended Analytical Methods to Evaluate Pretreatments

(1) The Starting Feedstock

We recommend that a detailed lignocellulosic analysis of the starting feedstock is carried out, including removal of the extractives and determination of their composition. Removal of the extractives is important since we have found that, if not removed, they can lead to elevated values for the lignin content. Furthermore, some biomass can contain significant amounts of water-soluble carbohydrates and extractives removal and characterisation will help to provide clarity on the source of the sugars found when the biomass sample is hydrolysed to determine its lignocellulosic composition. These water-soluble carbohydrates are also likely to be present in the liquid output of many pretreatment processes and, if their concentrations are not known, it may be incorrectly inferred that such sugars present in the liquid must come from lignocellulose.

Similarly, if you expect that there will be some starch in your sample then we also recommend that starch analysis of your starting feedstock be undertaken as this constituent may also be removed and hydrolysed in many pretreatment processes.

When determining the lignocellulosic composition of the sample it is important that each of the constituent sugars within the polysaccharides (e.g. glucose, xylose, arabinose, mannose, etc.) are measured separately so that their fate during the pretreatment process can be monitored.

Additionally, it may be useful to also determine the uronic acid composition of the sample.

(2) The Liquid Stream

We have found that much of the soluble carbohydrates in the liquid outputs of many pretreatment processes exist not as free sugars but as oligosaccharides that can contain as much as ten sugar units. For this reason, we would recommend that the liquid output is analysed for oligomeric sugars as well as for monosaccharides. We determine the oligomeric component by firstly analysing the original liquid for the free monosaccharides and disaccharides (e.g. cellobiose) in solution and then subjecting the liquid to a mild form of acid hydrolysis that will break apart any oligosaccharides into their constituent monosaccharide units. We then analyse the hydrolysate and the proportion of each sugar that is present in the original liquid in the oligomeric form can then be calculated by subtracting the pre-hydrolysis concentration from the post-hydrolysis concentration.

We would also advise that the liquid be analysed for the main organic acid and furanic sugar-degradation products.

analysis of biomass extracts at Celignis

(3) The Solid Residue

While it is likely that most of the conventional biomass extractives will have been removed in the pre-treatment, it is still necessary in many cases to undertake extractives removal of the solid residue from pre-treatment prior to undertaking its lignocellulosic analysis. This is because this residue may contain some of the liquid fraction sorbed onto its particles. The sugars and sugar-degradation products will be retained and concentrated on these particles if the sample is dried and may still be retained even if the sample is washed. Hence, if they are not removed prior to hydrolysis, these sugars may be assumed to come from lignocellulose, giving a false impression of the efficiency of the pre-treatment.

It is also possible that the composition of these sorbed water-soluble constituents will be different from that of the liquid fraction, so we recommend that the water extract is also characterised for its own monomeric/oligomeric sugar composition. These results, coupled with the data from the previous analyses, will help to give a clear and detailed picture on the fate of cellulose, hemicellulose, and lignin during the pretreatment process. We would also recommend that the ash content is determined to see how much ash is solubilised by the pre-treatment.

Analysis at Celignis

We have a lot of experience in analysing many products (both liquid and solid) from biomass pretreatment processes. These samples have covered a wide variety of starting feedstocks and pretreatment processes and conditions. We are able to undertake all of the analytical techniques described above and can provide data that can guide you in engineering the most appropriate pretreatment conditions for your feedstocks and processes.

Please contact us with your specific requirements and we will be happy to select a set of analysis packages suitable for your needs. There is also additional information on the website relating to our analytical techniques for pretreatment liquids, oligosaccharides, sugar-degradation products, and the solid residues from pretreatment processes.

Publications on Pre-treatment By The Celignis Team

Swart, L. J., Bedzo, O. K. K., van Rensburg, E., Gorgens, J. F. (2021) Intensification of Xylo-oligosaccharides Production by Hydrothermal Treatment of Brewers Spent Grains: The Use of Extremely Low Acid Catalyst for Reduction of Degradation Products Associated with High Solid Loading, Applied Biochemistry and Biotechnology 193: 1979-2003


Brewers' spent grains (BSG) make up to 85% of a brewery's solid waste, and is either sent to landfill or sold as cheap animal feed supplement. Xylo-oligosaccharides (XOS) obtained from BSG are antioxidants and prebiotics that can be used in food formulations as low-calorie sweeteners and texturisers. The effect of extremely low acid (ELA) catalysis in liquid hot water (LHW) hydrothermal treatment (HTT) was assessed using BSG with dry matter contents of 15% and 25%, achieved by dewatering using a screw press. Batch experiments at low acid loadings of 5, 12.5 and 20 mg/g dry mass and temperatures of 120, 150 and 170 C significantly affected XOS yield at both levels of dry mass considered. Maximum XOS yields of 76.4% (16.6 g/l) and 65.5% (31.7 g/l) were achieved from raw BSG and screw pressed BSG respectively, both at 170 C and using 5 mg acid/g dry mass, after 15 min and 5 min, respectively. These XOS yields were obtained with BSG containing up to 63% less water and temperatures more than 20 C lower than that reported previously. The finding confirms that ELA dosing in LHW HTT allows lowering of the required temperature that can result in a reduction of degradation products, which is especially relevant under high solid conditions. This substantial XOS production intensification through higher solid loadings in HTT not only achieved high product yield, but also provided benefits such as increased product concentrations and decreased process heat requirements.

Swart, L. J., Peterson, A. M., Bedzo, O. K. K., Gorgens, J. F. (2021) Techno-economic analysis of the valorization of brewers spent grains: production of xylitol and xylo-oligosaccharides, Journal of Chemical Technology & Biotechnology 96(6): 1632-1644


Brewers spent grains (BSG) represents around 85% of a brewery's solid waste and common disposal to landfill is increasingly more difficult. Yet BSG is a food-grade by-product with potential economic valorization that can significantly improve resource efficiency and reduction in carbon emissions. This study investigated valorization of BSG through the application of novel high solids hydrothermal processing technology in a small-scale biorefinery, annexed to a brewery. It focused on three scenarios for the production of: (A) the sugar replacement xylitol; (B) prebiotic xylo-oligosaccharide (XOS); and (C) co-production of xylitol and XOS. Economic assessment was conducted by comparing the capital and operating expenditure from process simulations created in Aspen Plus. The process models developed were supplemented with experimental data to improve accuracy.
Internal rate of return (IRR) values obtained were greater than the hurdle rate of 9.7% for all scenarios when considering a conservative market price for xylitol and XOS as US$4500 t-1, yet dedicated production of XOS was economically more favourable with a minimum required selling price (MRSP) of US$2509 t-1 compared to US$4153 t-1 for xylitol. Additionally, the scenario for co-production of xylitol and XOS achieved the lowest MRSP of US$2182 t-1. By-products significantly contributed to 32.7%, 14.2% and 27.5% of the revenue generated in scenarios A, B and C, respectively.
These results provide a good platform from which to develop the cost-effective commercial production of XOS and xylitol from BSG.

Bedzo, O. K. K., van Rensburg, E. and Gorgens, J. F. (2021) Investigating the effect of different inulin-rich substrate preparations from Jerusalem artichoke (Helianthus tuberosus L.) tubers on efficient inulooligosaccharides production, Preparative Biochemistry and Biotechnology 51(5): 440-449


Commercial production of inulooligosaccharides (IOS) relies largely on chicory roots. However, Jerusalem artichoke (JA) tubers provide a suitable alternative due to their high inulin content and low cultivation requirements. In this study, three inulin-rich substrate preparations from JA were investigated to maximize IOS production, namely powder from dried JA tuber slices (Substrate 1), solid residues after extracting protein from the JA powder (Substrate 2) and an inulin-rich fraction extracted from protein extraction residues (Substrate 3). The preferred temperature, pH and inulin substrate concentration were determined after which enzyme dosage and extraction time were optimized to maximize IOS extraction from the three substrates, using pure chicory inulin as benchmark. Under the optimal conditions, Substrate 3 resulted in the highest IOS yield of 82.3% (w/winulin). However, IOS production from the Substrate 1 proved more efficient since it renders the highest overall IOS yield (mass of IOS per mass of the starting biomass). In the case of co-production of protein and IOS from the JA tuber in a biorefinery concept, IOS production from the Substrate 2 is preferred since it reduces the inulin losses incurred during substrate preparation. For all the inulin-rich substrates studied, an enzyme dosage of 14.8 U/ginulin was found to be optimal at reaction time less than 6 h. JA tuber exhibited excellent potential for commercial production of IOS with improved yield and the possible advantage of a reduced biomass cost.

Bedzo, O. K. K., Dreyer, C. B., van Rensburg, E., Gorgens, J. F. (2021) Optimisation of Pretreatment Catalyst , Enzyme Cocktail and Solid Loading for Improved Ethanol Production from Sweet Sorghum Bagasse, BioEnergy Research


weet sorghum bagasse displays many characteristics rendering it a promising substrate for lignocellulosic ethanol production. In this study, the steam pretreatment catalyst, enzymatic hydrolysis and the substrate loading for the fermentation were investigated in order to maximise the production of ethanol from the feedstock. The results deemed water as a sufficient pretreatment catalyst since the SO2 impregnation of the biomass did not produce any significant beneficial effects on the yield of ethanol produced. The preferred pretreatment and enzymatic hydrolysis conditions were incorporated in a fed-batch simultaneous saccharification and fermentation (SSF) process using pressed-only (not washed) WIS at a final solid loading of 13% (w/w) that resulted in the targeted ethanol concentration of 39 g/L with a corresponding yield of 82% of the theoretical maximum. Yeast inhibition coupled with significant glucose accumulation was observed at higher solid loadings of 16% and 20%. Ultimately, the sweet sorghum bagasse could be integrated into existing ethanol production regimes to improve the global bioenergy production.

Bedzo, O. K. K., Mandegari, M. and Gorgens, J. F. (2020) Techno-economic analysis of inulooligosaccharides, protein, and biofuel co-production from Jerusalem artichoke tubers: A biorefinery approach, Biofuels Bioproducts & Biorefining-Biofpr 14(4): 776-793


Jerusalem artichoke (JA) is a crop with excellent potential for application in biorefineries. It can resist drought, pests, and diseases and can thrive well in marginal lands with little fertilizer application. The JA tubers contain considerable quantities of inulin, which is suitable for the production of inulooligosaccharides (IOS), as a high-value prebiotic, dietary fiber. In this study, five JA tuber biorefinery scenarios were simulated in Aspen Plus and further evaluated by techno-economic and sensitivity analyses. Production of IOS, proteins and animal feed was studied in scenarios A and C, applying various biorefinery configurations. Scenario B explored the option of producing only IOS and the sale of residues as animal feed. Scenarios D and E investigated the economic potential of biofuel generation from residues after IOS and protein production by generation of biogas and ethanol respectively, from residues. Based on the chosen economic indicators, scenario B resulted in the lowest minimum selling price (MSP) of 3.91 US$ kg-1 (market price 5.0 US$ kg-1) with correspondingly reduced total capital investment (TCI) and total operating cost (TOC) per mass unit produced of IOS of 18.91 and 2.59 US$ kg-1 respectively, compared with other studied scenarios. Considering the set production scale, it is more profitable when the residues are sold as animal feed instead of being converted into biofuel, due to the capital-intensive nature of the biofuel production processes. The coproduction of protein had a negative impact on the economics of the process as the associated capital and operating expenditure outweighed the associated revenue.

Gottumukkala L.D, Haigh K, Gorgens J (2017) Trends and advances in conversion of lignocellulosic biomass to biobutanol: microbes, bioprocesses and industrial viability, Renewable and Sustainable Energy Reviews 76: 963-973

Biobutanol has gained attention as an alternative renewable transportation fuel for its superior fuel properties and widespread applications in chemical industry, primarily as a solvent. Conventional butanol fermentation has drawbacks that include strain degeneration, end-product toxicity, by-product formation, low butanol concentrations and high substrate cost. The complexity of Clostridium physiology and close control between sporulation phase and ABE fermentation has made it demanding to develop industrially potent strains. In addition to the isolation and engineering of superior butanol producing bacteria, the development of advanced cost-effective technologies for butanol production from feedstock like lignocellulosic biomass has become the primary research focus. High process costs associated with complex feedstocks, product toxicity and low product concentrations are few of the several bioprocess challenges involved in biobutanol production. The article aims to assess the challenges in lignocellulosic biomass to biobutanol conversion and identify key process improvements that can make biobutanol commercially viable.

Parameswaran, B, Raveendran S, Singhania, R.R, Surender V, L Devi, Nagalakshmi S, Kurien N, Sukumaran R.K, Pandey A. (2010) Bioethanol production from rice straw: an overview, Bioresource technology 101(13): 4767-4774

Rice straw is an attractive lignocellulosic material for bioethanol production since it is one of the most abundant renewable resources. It has several characteristics, such as high cellulose and hemicelluloses content that can be readily hydrolyzed into fermentable sugars. But there occur several challenges and limitations in the process of converting rice straw to ethanol. The presence of high ash and silica content in rice straw makes it an inferior feedstock for ethanol production. One of the major challenges in developing technology for bioethanol production from rice straw is selection of an appropriate pretreatment technique. The choice of pretreatment methods plays an important role to increase the efficiency of enzymatic saccharification thereby making the whole process economically viable. The present review discusses the available technologies for bioethanol production using rice straw.